Association Of Arsenic Exposure During Pregnancy With Telomere Length And Telomere Attrition In Newborns And 3-year-old Children

Telomere is a DNA-protein complex located at the end of chromosomes in eukaryotic cells that undergo progressive attrition with each cell division.When the telomere length shortens to a critical value,it will trigger cell aging or apoptosis.Therefore,telomere length is an important biomarker reflecting the risk of aging and age-related diseases,and it is also an independent predictor of mortality.Telomere attrition occurs throughout the lifespan,and available studies suggest that telomere attrition is fastest in early life.Newborn telomere length and telomere attrition rate in early life are important factors determining telomere length in adulthood.Therefore,early life may be a critical window to assess the lifetime telomere length.However,studies on telomere length changes from birth to infancy are lacking,and the factors affecting age-related changes in telomere length early in life are unclear.Exposure to adverse environmental factors during pregnancy may have a long-term impact on disease susceptibility and longevity in adulthood by affecting telomere attrition rates early in life.Arsenic is an environmental pollutant that exists widely in the natural environment.Pregnant women and fetuses,as sensitive groups,are more vulnerable to arsenic exposure.Although epidemiological studies have explored the effect of arsenic exposure on telomere length,the results are not consistent.However,most of the current studies only measure the level of total arsenic and lack the assessment of different forms of arsenic and arsenic metabolism.In addition,genetic factors are also considered to play an essential role in determining individual telomere length.A series of genome-wide association studies（GWAS）have successively reported multiple genetic variants associated with telomere length.However,few cohort studies have reported genetic polymorphisms and longitudinal telomere length changes in early life.Therefore,based on a prospective birth cohort,this study explored the influencing factors of telomere length and telomere attrition in newborns and 3-year-old children from multiple dimensions of maternal and newborn sociodemographic characteristics,arsenic exposure during pregnancy,and genetic variation in telomere-related genes.And we further explored the interaction between arsenic exposure during pregnancy and genetic variation of telomere-related genes on telomere attrition.Part 1:Basic characteristics and influencing factors of telomere attrition n newborns and 3-year-old childrenObjective:To explore the basic characteristics and general influencing factors of telomere length and telomere attrition in newborns and 3-year-old children.Methods:This study was based on a birth cohort established at Wuhan’s Women’s and Children’s Health Care Center.The study included 879 mother-child pairs whose newborn cord blood and peripheral blood at 3-year-old were collected from 2014 to 2017.Information on general demographic characteristics of pregnant women and newborns,lifestyle habits during pregnancy,nutritional supplements during pregnancy,and complications during pregnancy were obtained by questionnaires and systematic medical records.The relative telomere length of leukocytes in cord blood of newborns and peripheral blood of 3-year-old children were measured by real-time quantitative PCR to assess the rate of telomere attrition over three years.A generalized linear regression model was used to explore the potential influencing factors of telomere length in newborn and 3-year-olds and telomere attrition rate over the three years.Results:The geometric mean of relative telomere length in newborn and 3-year-old children was 0.86 and 0.68（T/S value）.The relative telomere length of children decreased significantly over the three years（P<0.001）,and the median（interquartile range）telomere attrition rate over the three years was-5.12（-43.18,40.06）.The results of multivariate regression analysis showed that compared with the newborn of the lower mother’s education group（high school and below）,the telomere length of the newborn in the higher mother’s education group（high school and above）increased by 10.80%（95%CI:2.68%,19.56%;P=0.008）;compared with boy newborns,telomere length in girl newborns increased by 6.36%（95%CI:0.22%,12.88%;P=0.042）;compared with children whose mothers slept 7～9hours at night during pregnancy,the telomere attrition rate of children whose mothers slept less than 7 hours at night during pregnancy increased by 20.45%（95%CI:-40.04%,-0.87%;P=0.041）.Stratified analysis by sex found that compared with boys with normal pre-pregnancy body mass index（BMI）mothers（18.5 kg/m²≤BMI≤23.9 kg/m²）,the telomere attrition rate of boys whose mothers had a pre-pregnancy BMI≥24 kg/m² had increased by 19.38%（95%CI:-37.88%,-0.87%;P=0.040）;compared with boys whose mothers did not have gestational diabetes,the telomere attrition rate of boys with mothers had gestational diabetes had increased by 23.57%（95%CI:-46.39%,-0.75%;P=0.043）.No statistically significant associations were observed between other factors,telomere length in newborn and 3-year-old children,and telomere attrition rate over the three years（P>0.05）.Conclusions:This study found that the girls and mothers with higher education levels were significantly associated with the longer telomere length of newborns.The shorter nighttime sleep duration during pregnancy,overweight before pregnancy and gestational diabetes were significantly associated with increased telomere attrition rates in children over three years.The results provide new insights into the potential mechanisms linking maternal exposure to lifetime disease risk.Part 2:Association of arsenic exposure during pregnancy with telomere ength and telomere attrition in neonates and 3-year-old childrenObjective:To evaluate the urinary arsenic metabolite concentrations and arsenic metabolism efficiency of pregnant women in the first,second,and third trimesters of pregnancy and explore the association between arsenic exposure during pregnancy and telomere length and telomere attrition in newborns and 3-year-old children.Methods:The subjects were 645 mother-child pairs who provided urine samples in the first,second,and third trimesters in the first part of the study population.High performance liquid chromatography-Inductively coupled plasma-mass spectrometry（HPLC-ICP-MS）was used to detect five arsenic species in urine,such as arsenobetaine（As B）,arsenite(As³⁺),arsenate(As⁵⁺),monomethylarsonic acid（MMA）,and dimethylarsenic acid（DMA）,and calculate five arsenic metabolism efficiency indexes,such as inorganic arsenic percentage（i As%）,MMA percentage（MMA%）,MMA percentage（DMA%）,primary methylation index（PMI）and secondary methylation index（Secondary methylation index,SMI）.Generalized estimated equation（GEE）was used to analyze the association between repeated measurement of urinary arsenic metabolite concentration and metabolic efficiency in the first,second,and third trimesters and telomere length in newborns and 3-year-old children,and rates of telomere attrition in children over three years.Results:After adjusting for confounding factors,the telomere attrition rate of children over three years in the middle and highest trimester of MMA increased by 9.43%（95%CI:-18.49%,-0.37%）and 10.13%（95%CI:-19.40%,-0.85%）respectively,compared with the lowest trimester of urinary MMA in the second trimester.No significant associations were observed between concentrations of other urinary arsenic metabolites and rates of telomere attrition in children over three years.In addition,when the metabolic efficiency of urinary arsenic was divided into three groups according to tertile,compared with the lowest tertile group,the attrition rate of children over the three years in the highest tertile of MMA%in the first and second trimester,the highest tertile of i As%in the first trimester and the highest tertile of PMI in the second trimester increased by 12.49%（95%CI:-21.55%,-3.43%）,12.59%（95%CI:-21.70%,-3.48%）,10.02%（95%CI:-19.07%,-0.97%）and11.33%（95%CI:-20.51%,-2.14%）,respectively.While the attrition rate of children over the three years in the highest tertile of DMA%in the first trimester and the highest tertile of SMI in the three trimesters decreased by 9.61%（95%CI:0.59%,18.63%）,11.83%（95%CI:2.81%,20.85%）,9.43%（95%CI:0.43%,18.44）,respectively%)and 9.34%（95%CI:0.27%,18.40%）.For the newborn telomere length,compared with the lowest tertile of SMI,neonatal telomere length increased by 6.71%（95%CI:0.69%,13.08%）in the middle tertile of SMI in the first trimester.For the telomere length of 3-year-old children,compared with the lowest tertile of SMI,the telomere length of 3-year-old children in the middle and highest tertile of SMI in the first trimester and the highest tertile of SMI in the third trimester were respectively Increased by 14.40%（95%CI:2.78%,27.33%）,14.80%（95%CI:3.08%,27.85%）and 17.01%（95%CI:5.05%,30.34%）.The results of the gender-stratified analysis showed that the associations between MMA%,i As%,DMA%and SMI during pregnancy and telomere attrition rate were more significant in boys but not in girls.No statistically significant associations were observed between urinary arsenic metabolite concentrations and telomere length in newborn and 3-year-old children.Conclusions:Higher levels of MMA exposure in the second trimester were associated with increased telomere attrition rates in children over three years.Higher MMA%,i As%,and PMI during pregnancy were also associated with increased telomere attrition rates in children over three years.Lower DMA%and SMI were associated with shorter telomere length in newborns and 3-year-old children and telomere attrition rates in children over three years.These suggest that arsenic exposure during pregnancy and its lower metabolic efficiency may affect telomere length and telomere attrition rate in early life.Part 3:Interaction of arsenic exposure during pregnancy and telomere-related gene polymorphisms in association with telomere attrition in hildrenObjective:To investigate the effect of interaction between arsenic exposure during pregnancy and telomere-related gene polymorphisms on telomere attrition in children.Methods:The subjects of this part were 346 pairs of mothers and children with umbilical cord blood DNA samples in the second part,and the Illumina Human Asian Screening Array（ASA）gene chip was used to conduct whole genotyping of cord blood DNA.The GWAS catalog database,gene cards database,Pub Med and Google Scolar were used to search the 27 genes related to telomere length as candidate genes.From the typing results after quality control,152 single nucleotide polymorphisms（SNPs）were extracted from 27 gene regions.First,the generalized linear regression model was used to analyze the association between SNPs in the co-dominant,dominant,recessive,additive genetic models and the telomere attrition rate in children over three years.For SNPs significantly associated with telomere attrition rate(P_FDR<0.05),the linkage disequilibrium（LD）coefficient r2 was calculated and screened for independent SNPs（r²<0.8 and D’<0.8）using Haplo View 4.2software.The Genotype-Tissue Expression（GTEx）database was then used to analyze the gene expression regulatory functions of the SNPs,screen the Expression quantitative trait loci（e QTL）,and combine with literature reports to filter the final target genetic variants.Second-trimester urinary MMA concentrations were converted into categorical variables according to tertiles.Generalized linear regression models were used to analyze the association between urinary MMA concentrations and target SNPs in interaction and association with telomere attrition rate in children over three years.Results:Based on the four genetic models,58 SNPs were significantly associated with three-year telomere attrition rates in children(P_FDR<0.05).For the above 58 SNPs,based on the results of LD analysis and e QTL analysis and combined with literature reports,9target genetic variants（ACYP2-rs11125529,ACYP2-rs843719,OGG1-rs1052133,SENP7-rs939443,TERT-rs2736100,CARMIL1-rs10456324,RFWD3-rs4888262,ERCC1-rs3212961 and RTEL1-rs6010620）were selected for the subsequent interaction analysis.The interaction analysis results showed a significant interaction effect between the maternal urinary concentration of MMA in the second trimester and OGG1-rs1052133(P_int<0.05).After the rs1052133 genotype was stratified according to the dominant model,only in children with rs1052133 GG genotype was found that a significant association between the concentration of urinary MMA in the second trimester of pregnancy and the children’s three-year telomere attrition rate(P_trend=0.009).Among the rs1052133 GG genotype carriers,the telomere attrition rate in the highest tertile group increased by 72.43%（95%CI:-122.82%,-22.05%）compared with the lowest tertile of urinary MMA in the second trimester.There was no significant association between urinary MMA and telomere attrition rate in children with rs1052133 CG+CC genotype.In addition,the combined effect analysis results showed that compared with children in the lowest tertile of urinary MMAconcentration in the second trimester and with the rs1052133 GG genotype,children with the highest tertile of urinary MMA concentration in the second trimester and with the rs1052133 CC had a 42.26%（95%CI:-72.45%,-12.07%;P<0.05）increase in telomere attrition over three years,and the rate of telomere attrition increased by 26.39%（95%CI:-52.10%,-0.67%;P<0.05）in children with the middle tertile of urinary MMA concentration in the second trimester who carried the rs1052133 CG+GG genotype.Conclusion:There was a significant interaction effect between the rs1052133 located in the exonic region of the OGG1 gene and maternal urinary concentration of MMA in the second trimester associated with the rate of telomere attrition in children over three years.The negative association between urinary concentration of MMA in the second trimester and the telomere attrition rate was more pronounced in children carrying rs62035769 CC genotype.